Project 1 (Massagu): Mediators of lung adenocarcinoma metastasis PROJECT SUMMARY Our goal in this project is to elucidate the molecular and cellular determinants of lung adenocarcinoma (adenoCa) metastasis, in order to improve the prevention and treatment of lung cancer relapse. Nearly half of early-diagnosed (stage I and II) lung adenocarcinoma cases develop local and distant relapse despite surgical resection of the primary tumor. Although cancer cell entry into the circulation and extravasation into distant organs are important early steps in the metastatic cascade, the predominant concern in the clinic is about preventing and treating relapse that is driven by cancer cells that were disseminated since before diagnosis. Therefore, our focus is on metastatic colonization, including the phases of metastatic seeding, latency, and outbreak. Building on our previous and new mouse models of brain and multi-organ metastasis, in the current grant period we have identified mediators of metastatic latency and immune evasion by metastatic stem-like cells (MetSCs) (Malladi et al Cell 2016), and mediators of relapse by residual disease after erlotinib and crizotinib treatment of lung adenoCa (Obenauf et al Nature 2015). We also identified serpin mediators of lung MetSC survival in the brain, and defined L1CAM as a mediator of vascular cooption for metastatic outgrowth (Valiente et al Cell 2014). Moreover, we found that carcinoma-astrocyte gap junctions promote brain metastasis by cGAS-STING pathway transfer and pharmacologic modulation of gap junctions is effective against established brain lesions in these models (Chen et al Nature 2016). Our work translated into two clinical trials, one with gap junction modulator meclofenamate and the other anti-TNF antibody certolizumab, in patients with stage IV lung adenoCa. Our future Aim 1 is to define and target the role of L1CAM signaling in lung adenoCa metastasis. We will determine the origin of L1CAM+ lung adenoCa MetSCs, the role of L1CAM signaling in outgrowth, and how to target L1CAM for inhibition of metastasis initiation and propagation. Aim 2 is to elucidate the signaling dynamics and immune evasive state of micrometastatic disease. We will investigate the role of three key pathways ?WNT, TGF-? and Hippo? during MetSCs entry and exit from latency, and the role of NK and CD8+ T cells in enforcing latency by NKG2D-mediated recognition of ULBP ligands in MetSCs; the ultimate goal is to trigger immune-mediated clearance of residual disease. Our Aim 3 is to functionally define our recently identified mediators of CNS metastasis as therapeutic targets. We will characterize connexin 43, complement factor 3, and lipocalin 2 as mediators of brain and leptomeningeal metastasis, and will pre-clinically test pharmacologic inhibitors of these targets in combination with therapeutic approaches being developed by other members of this Program Project. Through this work we hope to contribute innovative ideas for the prevention and treatment of lung cancer relapse while retaining our long-term focus on discovering basic principles of metastasis.